Monitoring the dynamic of bacterial community and nitrogen cycle functional genes
expression during a N2O emission peak.
N. Theodorakopoulos*1, F. Degrune1,3, M. Lognoul2, D. Regaert2, B. Heinesch2, B. Bodson4, M. Aubinet2, M. Vandenbol1
[email protected]
1Microbiology and Genomics, Gembloux Agro-Bio tech, University of Liège, Avenue Maréchal Juin, 6, 5030 Gembloux (Belgium)
2Exchanges Ecosystems – Atmosphere, Department of Biosystem Engineering (BioSE), Gembloux Agro-Bio Tech, University of Liege
3AgricultureIsLife, Gembloux Agro-Bio tech, University of Liège, Passage des déportés, 2, 5030 Gembloux (Belgium)
4Crop Sciences, Gembloux Agro-Bio tech, University of Liège, Passage des déportés, 2, 5030 Gembloux (Belgium)
Environmental context
Among the main greenhouse gases (GHG), nitrous oxide (N2O) causes a serious environmental problem because of its global warming potential which is 298 times higher
than CO2 and because of its lifetime of 114 years. It is well known that microorganisms play an essential role in N2O emissions (through nitrification and denitrification) and
that agricultural soils emit most of this GHG. Thus, characterizing the dynamic of bacterial community and the expression of nitrogen cycle functional genes
during a N2O emission peak is of great interest to understand and anticipate N2O emissions and improve good agricultural practices recommendations.
Methods
Fig. 1 Automated closed-dynamicchamber system on the field
•
•
•
•
Automated closed-dynamic-chamber (Fig. 1) system was used to Record N2O emissions- 1 flux each 30 minutes (Fig. 2).
Soil samples (10 top cm) were collected at strategic time (in triplicates) (green circles).
Quantitative PCR was used to quantify gene expression during the observed peak (Fig. 3).
Massive sequencing of 16S rRNA gene (Ion Torrent, MOTHUR) was conducted to assess the bacterial community dynamic (Fig. 4).
Results
Fig. 2 N2O emissions and soil moisture during the experiment
Nitrous oxide fluxes determined with either a linear (empty triangle) or an exponential fit (filled triangle)
Microbiological sampling date and calculated fluxes means at the sampling time
Soil moisture (%)
Soil sampling over
time (8 dates) and
RNA extraction (fig. 2)
200
Rainfall
150
N2O emission peak
100
50
0
13/06
cDNA
15/06
17/06
19/06
1011
16SrRNA: bacterial 16SrRNA gene
expression was quantified to report
the global bacterial activity during
N2O emission peak.
1010
107
nirS: Nitrite reductase
(NO2- -> NO)
106
nirK: Nitrite reductase
(NO2- ->NO)
nifH: Nitrogenase reductase
(N2 -> NH4+)
105
nosZ: Nitrous oxide reductase
(N2O -> N2)
amoA: Ammonia monooxygenase
(NH4+ -> NH2OH)
**
104
103
17/07
19/07
21/07
23/06
25/06
27/06
(2) Microbial communities dynamic
was assessed using massive
sequencing (Ion Torrent)
23/07
25/07
27/07
Fig. 4 List of OTU positively and negatively correlated with nitrous oxide
Quantification of genes transcript (copies number per µg RNA)
Fig.3 Gene transcripts abundance evaluation by quantitative PCR
Significant differences are marked with *
(1) Quantitative PCR
analysis on N-genes cycle
and 16S rRNA genes
21/06
29/06
1/07
45
40
35
30
25
20
15
10
5
0
3/07
soil moisture (%)
ng N2O.N m-2 s-1
250
Relationship between 16SrRNA transcripts
abundance and the average of N2O emissions
(for each operational taxonomic unit (OTU)
phylum
class
order
family
genus
Proteobacteria
Proteobacteria
Proteobacteria
Proteobacteria
Proteobacteria
Proteobacteria
Proteobacteria
Proteobacteria
Proteobacteria
Proteobacteria
Proteobacteria
Proteobacteria
Proteobacteria
Proteobacteria
Proteobacteria
Proteobacteria
Proteobacteria
Proteobacteria
Actinobacteria
Proteobacteria
Proteobacteria
Proteobacteria
Proteobacteria
Nitrospirae
Proteobacteria
Firmicutes
Actinobacteria
Proteobacteria
Chloroflexi
Bacteroidetes
Acidobacteria
Firmicutes
Actinobacteria
Nitrospirae
Actinobacteria
Chloroflexi
Betaproteobacteria
Betaproteobacteria
Betaproteobacteria
Betaproteobacteria
Deltaproteobacteria
Betaproteobacteria
Alphaproteobacteria
Betaproteobacteria
Alphaproteobacteria
Betaproteobacteria
Alphaproteobacteria
Alphaproteobacteria
Alphaproteobacteria
Alphaproteobacteria
Alphaproteobacteria
Alphaproteobacteria
Alphaproteobacteria
Alphaproteobacteria
Actinobacteria
Alphaproteobacteria
Alphaproteobacteria
Alphaproteobacteria
Deltaproteobacteria
Nitrospira
Alphaproteobacteria
Bacilli
Actinobacteria
Alphaproteobacteria
Chloroflexia
Sphingobacteriia
Acidobacteria
Bacilli
Actinobacteria
Nitrospira
Actinobacteria
Chloroflexia
Burkholderiales
Burkholderiales
Nitrosomonadales
Burkholderiales
Myxococcales
Burkholderiales
Caulobacterales
unclassified
Caulobacterales
Burkholderiales
Rhizobiales
Caulobacterales
Rhizobiales
Rhizobiales
Rhodospirillales
Rhizobiales
Caulobacterales
Rhizobiales
Micromonosporales
Rhizobiales
Rhizobiales
Rhizobiales
Myxococcales
Nitrospirales
Caulobacterales
Bacillales
Streptomycetales
Rhodospirillales
Chloroflexales
Sphingobacteriales
Acidobacteriales
Bacillales
Streptosporangiales
Nitrospirales
Streptomycetales
Chloroflexales
Comamonadaceae
unclassified
Nitrosomonadaceae
Comamonadaceae
Sandaracinaceae
Comamonadaceae
Caulobacteraceae
unclassified
Caulobacteraceae
Comamonadaceae
Rhizobiales_Incertae_Sedis
Caulobacteraceae
Rhizobiales_Incertae_Sedis
Hyphomicrobiaceae
DA111
Rhizobiaceae
Caulobacteraceae
unclassified
Micromonosporaceae
Bradyrhizobiaceae
Rhizobiaceae
Rhizobiaceae
Phaselicystidaceae
Nitrospiraceae
Caulobacteraceae
Paenibacillaceae
Streptomycetaceae
Rhodospirillaceae
Roseiflexaceae
Chitinophagaceae
Acidobacteriaceae_(Subgroup_1)
Bacillaceae
Streptosporangiaceae
Nitrospiraceae
Streptomycetaceae
Roseiflexaceae
Polaromonas
unclassified
unclassified
Albidiferax
Sandaracinus
unclassified
Phenylobacterium
unclassified
Phenylobacterium
unclassified
Rhizomicrobium
Phenylobacterium
Nordella
Hyphomicrobium
unclassified
unclassified
Caulobacter
unclassified
unclassified
unclassified
Rhizobium
unclassified
Phaselicystis
Nitrospira
Phenylobacterium
Paenibacillus
Streptomyces
Skermanella
Roseiflexus
Flavisolibacter
unclassified
Bacillus
Streptosporangium
Nitrospira
Streptomyces
Roseiflexus
coefficient
correlation
-0,41
-0,41
-0,41
-0,42
-0,42
-0,42
-0,42
-0,43
-0,44
-0,55
0,54
0,53
0,53
0,52
0,50
0,50
0,49
0,49
0,49
0,48
0,47
0,47
0,47
0,46
0,46
0,45
0,45
0,44
0,44
0,44
0,43
0,42
0,42
0,42
0,41
0,40
-
+
Transcript quantity of nirS, nirK, nifH, nosZ, and 16srRNA genes showed no significant
On the 14267 OTUs identified in this experiment, only 36 showed an activity significantly
changes during the N2O emission peak. The transcript quantity of amoA gene showed a significant
change positively correlated with N2O emissions. amoA gene encodes for the ammonia
correlated with N2O emission (>0,40, <-0,40, n=24). Among them, 2 OTUs positively
correlated were identified as members of Nitrospira genus which is responsible of the
nitrification. One OTU affiliated to the family Nitrosomonadaceae was negatively
correlated with N2O emissions. Members of this family have generally the gene amoA.
monooxygenase which catalyzes during the nitrification the oxidation of ammonium to
Conclusions
hydroxylamine (NH2OH). NH2OH can subsequently be abiotically transformed in N2O process.
• The use of automated closed-dynamic-chamber system allowed the determination of N2O emissions at a fine scale.
• Denitrification genes expression abundance did not significantly evolve during N2O emissions.
• Nitrification marker (amoA gene) showed a significant correlation with N2O emissions. amoA gene expression appeared to be the best proxy to follow N2O
emissions (R² = 0,89). amoA positive correlation wasn’t explained by an increase of Nitrosomonadaceae members and could therefore be the result of a gene induction .
• Bacterial community structure remained globally stable except for 36 OTUs which showed a positive or negative significant correlation with N2O emissions
•
(including members of the nitrification process).
Denitrification was expected after the rainfall but results demonstrated that nitrification could be the main driver of N2O emissions in this agricultural soil.